Contribution of phosphodiesterase isoenzymes and cyclic nucleotide efflux to the regulation of cyclic GMP levels in aortic smooth muscle cells.

Involvement of phosphodiesterase isoenzymes (PDEs) in guanosine-3',5'-cyclic monophosphate (cGMP) hydrolysis was analyzed in aortic smooth muscle cells. Four families of PDEs were separated from pig aorta: PDE1 (calcium-calmodulin-activated), PDE3 (cGMP-inhibited), PDE4 (adenosine 3',5'-cyclic monophosphate [cAMP]-specific), and PDE5 (cGMP-specific). Within this PDE complement, PDE1 and PDE5 mostly contributed to the hydrolysis of cGMP both in the presence and absence of calcium-calmodulin. The role of these isoenzymes in cGMP degradation was analyzed in primary cultures of porcine aortic smooth muscle cells after stimulation with sodium nitroprusside (SNP) or atrial natriuretic factor (ANF). Pretreatment with 10 microM zaprinast, a concentration that selectively inhibits PDE5, did not potentiate the SNP- or ANF-induced rise of cGMP, questioning the widespread opinion that only PDE5 accounts for cGMP hydrolysis in this tissue. Further evidence came from experiments assessing the effect of zaprinast or 3-isobutyl-1-methylxanthine at concentrations inhibiting both type 1 and type 5 isoenzymes, in which this potentiation was clearly seen. Contribution of cGMP egression to the control of intracellular cGMP levels after SNP or ANF stimulation was also investigated. Shortly after guanylate cyclase activation, extracellular cGMP levels surpassed intracellular levels. However, comparison of the amounts of cGMP extruded to the extracellular medium with those degraded by PDEs leads to the conclusion that efflux is of relatively minor importance in regulating intracellular cGMP levels. In cells made tolerant to SNP, selective PDE5 inhibition synergistically increased intra- and extracellular cGMP amounts after SNP stimulation. These results indicate a previously undescribed greater relevance of PDE5 after tolerance development in aortic smooth muscle cells.     

Interactions between isoprenaline, sodium nitroprusside, and isozyme-selective phosphodiesterase inhibitors on ADP-induced aggregation and cyclic nucleotide levels in human platelets.

The interactions between isoprenaline, sodium nitroprusside, and the isozyme-selective phosphodiesterase inhibitors OPC 3911 ("cAMP specific") and zaprinast ("cGMP specific") on platelet aggregation induced by adenosine diphosphate (ADP) and on levels of cAMP and cGMP were studied. Isoprenaline at 10(-6)M diminished aggregation by 28%, and this effect was enhanced by 10(-7)-10(-6)M OPC 3911. Neither 10(-6) M isoprenaline nor 10(-7)M OPC 3911 elevated cAMP, but in combination they caused a significant rise in cAMP (27% above the basal level), accompanying the synergistic functional inhibition, without affecting cGMP levels. Sodium nitroprusside at 10(-5) M diminished aggregation by 39%, elevated cGMP levels (81-110%), but also caused a statistically significant increase in cAMP (21-32%), and enhanced the effects of 10(-6)M isoprenaline on cAMP levels. Zaprinast at 10(-5) M caused a modest inhibition of aggregation by 20%, and a small increase in cGMP (20%), and it clearly enhanced the effects of 10(-5)M sodium nitroprusside on both cGMP and cAMP levels, but not on aggregation. The cAMP-increasing effect of sodium nitroprusside might be a consequence of a cGMP-mediated inhibition of the "low-Km cGMP-inhibited phosphodiesterase" that is also inhibited by OPC 3911. The effects of all of the drugs on ADP-induced aggregation seem to depend more on their effect on cAMP levels than on the levels on cGMP.     

Role of cyclic AMP- and cyclic GMP-phosphodiesterases in the control of cyclic nucleotide levels and smooth muscle tone in rat isolated aorta. A study with selective inhibitors

Three isoforms of cyclic nucleotide phosphodiesterase (PDE) have been recently isolated from aortic tissue and two of them specifically hydrolyzed adenosine 3',5'-cyclic monophosphate (cAMP) and guanosine 3':5'-cyclic monophosphate (cGMP), respectively (Lugnier et al., Biochem. Pharmac. 35, 1743, 1986). The role of these forms in controlling cyclic nucleotide levels and smooth muscle tone was investigated by the use of PDE inhibitors. The effects of selective inhibitors of the two forms specifically hydrolyzing cAMP or cGMP (cAMP-PDE and cGMP-PDE, respectively) were compared to those of non-selective inhibitors of the three aortic PDE forms, including the calmodulin-sensitive one (CaM-PDE). Relaxation responses and accumulation of tissue cAMP and cGMP induced by these drugs were studied in precontracted rat isolated aorta, and compared to the effects of isoprenaline and forskolin (stimulants of adenylate cyclase) or sodium nitroprusside (SNP) and sodium azide (stimulants of guanylate cyclase). The eight PDE inhibitors tested all relaxed aorta with potencies that correlated with their potencies as inhibitors of cAMP-PDE, but not of cGMP-PDE. At a concentration producing half-maximal relaxation, all PDE inhibitors induced a moderate but significant accumulation of cAMP, which was comparable to the accumulation of cAMP elicited by half-maximally relaxing concentrations of adenylate cyclase stimulating agents. At this concentration, some PDE inhibitors (M&B 22,948, dipyridamole and to a lesser extent, trequinsin) also induced a significant increase in cGMP levels, of the same order of magnitude as that caused by agents stimulating guanylate cyclase. However, the cGMP-increasing effect of these inhibitors was dissociated from their relaxing effect. In particular, the relaxing concentrations of M&B 22,948 (a selective inhibitor of cGMP-PDE) were clearly higher than the cGMP-increasing concentrations of the compound. At a concentration at which they elicited 10% relaxation by themselves, the selective cAMP-PDE inhibitor, rolipram, as well as the mixed inhibitor of cAMP- and cGMP-PDE, AAL 05 (a cilostamide analogue) enhanced both the cAMP-increasing and the relaxing effect of isoprenaline. Under the same conditions, no clear enhancement of the relaxation induced by SNP was observed. Only M&B 22,948 showed a slight potentiating effect on SNP-induced relaxation, but this effect was limited to low concentrations of SNP (less than 10 nM).(ABSTRACT TRUNCATED AT 400 WORDS)     

The effect of unsaturated fatty acids on sodium nitroprusside stimulation of guanylate cyclase in the human astrocytoma clone, D384, and the human neuroblastoma clone, NB1-G

Sodium nitroprusside (SNP) stimulates cGMP formation to a greater extent in 20,000 g supernatant fractions of the human neuroblastoma clones NB1-G and SH-SY5Y than in the human astrocytoma clone D384. This suggests that these cell lines contain the soluble form of guanylate cyclase. Arachidonic, 8,11,14- and 11,14,17-eicosatrienoic acids inhibit SNP (10(-4) M)-stimulated cGMP formation more potently than the C18 unsaturated fatty acids linolenic and linoleic acids in D384 and NB1-G. In contrast the C20 saturated fatty acid, arachidic acid had little effect even at 10(-4) M concentration. In addition arachidonic and 8,11,14-eicosatrienoic acids inhibited basal guanylate cyclase activity, in NB1-G, over the same concentration range as they inhibited SNP-stimulated cGMP formation. No evidence could be obtained for the stimulation of guanylate cyclase by arachidonic acid in either NB1-G or D384. These results provide further support for suggestions that arachidonic acid or its metabolites may be important regulators of cGMP formation in the nervous system.     

Relation of cyclic nucleotide ratios to ischemic and reperfusion injury in nitric oxide-donor treated rat hearts

Nitric oxide (NO) donors given during ischemia possibly protect the myocardium by increasing tissue cyclic guanosine monophosphate (cGMP) and decreasing cytosolic Ca2+ levels. However, NO donors also elevate ischemic cyclic adenosine monophosphate (cAMP) levels, which exacerbates ischemic-reperfusion injury. The authors propose that suppression of this NO donor-induced increase in cAMP would improve the cardioprotective properties of these compounds. Langendorff perfused rat hearts were treated with sodium nitroprusside (SNP, 0.1 mM ) or glyceryl trinitrate (GTN, 1.0 microM ) and/or adenylyl cyclase (SQ, 50 microM ) or guanylyl cyclase (ODQ, 30-300 microM ) inhibitors during 40-min low-flow (0.2 ml/min) ischemia. Control reperfusion rate-pressure product (RPP) recoveries were 47 +/- 3% (n = 9) and improved to 59 +/- 1% (n = 11) (p < 0.05) with SNP treatment. Ischemic ODQ treatment decreased RPP recovery to 33 +/- 3% (n = 10) (p < 0.05). ODQ eliminated the cardioprotective effects of SNP (RPP recovery: 40 +/- 5% [n = 7] vs. 59 +/- 1% [p < 0.05]). Adenylyl cyclase inhibition improved RPP recovery from 59 +/- 1% (SNP) to 72 +/- 4% (SNP + SQ) (n = 11) (p < 0.05). The authors conclude that (a) suppression of the NO donor-induced elevations in ischemic cGMP levels (ODQ) worsened reperfusion RPP, (b) suppression of the NO donor-induced elevation in ischemic cAMP levels (SQ) further improved reperfusion RPP in NO donor-treated hearts, and (c) the severity of ischemic-reperfusion injury in the NO donor-treated heart was inversely related to ischemic-tissue cGMP levels and often directly related to the ischemic-tissue cAMP-to-cGMP ratio.     

Regulation of cAMP metabolism in mouse parotid gland by cGMP and calcium.

The interaction of hormones acting via the mobilization of calcium and stimulation of cAMP levels in cells was examined by determining the effects of carbachol and forskolin on cAMP and cGMP accumulation in mouse parotid gland. Treatment of isolated acini with either carbachol (0.01 to 20 microM) or forskolin (1 microM) alone produced little or no increase in cAMP levels; carbachol, however, augmented the effect of forskolin on cAMP accumulation approximately 3- to 4-fold. The effects of carbachol on forskolin-stimulated cAMP levels were further augmented approximately 10-fold in the presence of the phosphodiesterase inhibitor 3-isobutyl-1-methylxanthine (MIX) but not in the presence of "low Km" cGMP-inhibited phosphodiesterase inhibitor milrinone. Augmentation of cAMP levels also occurred in the presence of carbachol plus the beta-adrenergic agonist isoproterenol (0.01 microM). In either the presence or absence of forskolin, carbachol increased cGMP levels independently of the inclusion of MIX and in a fashion parallel to that observed for cAMP accumulation. In the presence of forskolin (1 microM), the concentration of carbachol that produced half-maximal effects on cAMP and cGMP levels was 0.62 and 0.72 microM, respectively. Similar values were obtained in the presence of MIX. Cyclic GMP levels were also enhanced by carbachol plus isoproterenol. Hydroxylamine, as well as dibutyryl-cGMP and 8-bromo-cGMP in combination with forskolin, mimicked the effects of carbachol plus forskolin on cAMP levels. LY83583 (6-anillino-5,8-quinolinedione), an agent that lowers cGMP by inhibiting guanylate cyclase, reduced basal levels of cGMP and also completely prevented the increase in cGMP caused by carbachol plus forskolin. In these experiments, however, the augmentation of forskolin-stimulated cAMP levels by carbachol was reduced by approximately 50%. Additional studies suggest that calcium is also required for carbachol augmentation of forskolin-stimulated cAMP accumulation by effects on the adenylate cyclase complex. Augmentation of cAMP levels by carbachol did not involve effects on cAMP degradation. The results suggest that, when cAMP synthesis is stimulated by forskolin or isoproterenol, the muscarinic agonist carbachol augments cAMP accumulation by mechanisms involving cGMP and calcium in mouse parotid gland.     

Effect of vasodilators, including nitric oxide, on the release of cGMP and cAMP in the isolated perfused rat kidney

In isolated Tyrode-perfused rat kidneys, the release of the cyclic nucleotides cAMP and cGMP was measured in response to several vasodilators, including nitric oxide (NO). During vasoconstrictions induced by methoxamine, a basal release of both cyclic nucleotides was detected in the renal effluent (357 ± 32 fmol/min for cGMP and 3097 ± 219 fmol/min for cAMP). Injection of acetylcholine (ACh; 11 nmol), sodium nitroprusside (SNP; 0.8 nmol) and atrial natriuretic factor (ANF; 80 pmol) caused a marked release of cGMP. The cGMP release induced by ACh was not altered by indomethacin (3 μM) but was markedly reduced by the NO synthase inhibitor nitro-L-arginine (L-NNA; 200 μM). Authentic NO (0.16–80 nmol) caused dose-dependent vasodilatations that were accompanied by increases in the overflow of cGMP. The vasodilatations caused by forskolin (6 nmol) and prostacyclin (PGI₂; 3–52 nmol) were not accompanied by an overflow of cGMP. The vasodilator responses to 5-hydroxytryptamine (5-HT; 0.25–2 μmol), obtained in presence of the 5-HT₂ receptor blocker ritanserin (10 nM) and the 5-HT₃ blocker ICS 205930 (10 nM), were markedly reduced by L-NNA; however, they were not accompanied by the renal release of cGMP. Both forskolin and PGI₂ induced the release of cAMP from perfused rat kidneys; ACh, 5-HT and 5-carboxamidotryptamine (5-CT) also evoked a significant release of cAMP into the renal effluent. The release of cAMP induced by ACh and 5-HT was reduced by indomethacin and L-NNA. Higher doses of NO released cAMP from the perfused rat kidneys. Our data illustrate that both cAMP and cGMP can be released by vasodilator substances into the venous effluent of isolated perfused rat kidneys. The dilator responses to 5-HT were sensitive to the NO synthase inhibitor L-NNA and were accompanied by the release of cAMP and not by the release of cGMP. Our data suggest that the dilator responses may be due to NO released from endothelial cells, which then activates adenylyl cyclase either directly or indirectly.     

On the role of cyclic nucleotides in the regulation of cardiac contractility and glycolysis during hypoxia

A possible involvement of cyclic nucleotides (cAMP and cGMP) in the regulation of cardiac contractility and glycolysis during hypoxia was examined in spontaneously beating rat atria. A reduction of the high oxygen saturation (HiOxSa) of the incubation medium from 95-100% to half produced a rapid decline of the amplitude. The deterioration of 50% was seen after 30 sec. of hypoxia. The decline was partly antagonized by noradrenaline (NA, 1 X 10(-6) M) or hypercalcaemia (5.7 X 10(-3) M instead of 1.9 X 10(-3) M). The cAMP level remained unchanged during the first 12 min. of hypoxia, but the cGMP content increased gradually and reached a significantly increased level in 4-8 min. Paradoxically, the production of lactate decreased, after 30 sec. of hypoxia, but accelerated then 2-4 min. after the onset of hypoxia. The depletion of creatine phosphate and ATP stores was initiated after 2 min. of hypoxia. The arterial content of the active forms of phosphofructokinase and lactate dehydrogenase gradually rose during hypoxia. Sodium nitroprusside (SNP, 1 X 10(-4) M) and NA produced increases in cGMP and cAMP levels, respectively, both in HiOxSa and hypoxia. SNP induced a slight and NA a marked increase in the amplitude in HiOxSa. Verapamil (1 X 10(-6) M) decreased the contractility, but did not affect the levels of cAMP and cGMP. Both SNP and verapamil decreased the lactate production, but they could not resist the NA-induced increase in the atrial lactate level. Hypercalcaemia increased the amplitude but slightly reduced the lactate production in HiOxSa. 45Ca-uptake was reduced to about 35 per cent of control as measured between 5 and 10 min. of hypoxia. It is concluded that the lack of oxygen could have direct and parallel effects on the sarcolemma and on the mitochondria. The former could result in the deterioration of contractility and the latter in the termination of aerobic energy production. Cyclic nucleotides are not involved in either of these phenomena. However, at the low rate of anaerobic glycolysis, e.g. in HiOxSa or at the very early stage of hypoxia, cGMP could inhibit and cAMP accelerate the lactate production.     

Diphenylamine-2-carboxylic acid potentiates the cyclic nucleotides-mediated relaxation of porcine coronary artery: possible involvement of the inhibitory effect on the efflux of cyclic nucleotides

We examined the effect of diphenylamine-2-carboxylic acid (DPC), which has been shown to inhibit the efflux of cyclic nucleotides from vascular smooth-muscle cells, on the relaxant responses to forskolin, an adenylyl cyclase activator, and sodium nitroprusside (SNP), an NO donor, in the porcine coronary arteries. DPC (100 microM), which caused only a minor effect by itself, significantly augmented the relaxant responses to forskolin and SNP in the preparations contracted with 30 mM KCl. On the other hand, DPC did not affect the relaxant responses to nifedipine and cromakalim. Forskolin (10 microM) induced an accumulation of adenosine 3', 5'-cyclic monophosphate (cAMP) in the porcine coronary arteries, which was associated with an accumulation of cAMP in the incubation media. The intracellular cAMP response to forskolin was enhanced by DPC, whereas the extracellular cAMP response was reduced. The effects of SNP on guanosine 3', 5'-cyclic monophosphate (cGMP) accumulation were examined in the presence of 3-isobutyl-l-methylxanthine (500 microM) because cGMP was not found in the tissue and the incubation medium in the absence of the phosphodiesterase inhibitor. DPC significantly decreased the SNP-induced release of cGMP to the extracellular space, whereas it did not affect the accumulation of cGMP in the tissue. These results suggest that DPC inhibits the efflux of cyclic nucleotides. It is likely that the inhibitory effect of DPC on cAMP efflux contributes to the enhancement of tissue cAMP accumulation and relaxation produced by the agents that activate adenylyl cyclase. Thus, the transport system(s) of cyclic nucleotides may be a novel target for the prevention and/or treatment of various cardiovascular diseases.     

Relaxation and potentiation of cGMP-mediated response by ibudilast in bovine tracheal smooth muscle

The effects of ibudilast, an inhibitor of phosphodiesterases (PDEs), on tension, levels of guanosine 3',5'-cyclic monophosphate (cGMP) and adenosine 3',5'-cyclic monophosphate (cAMP) were investigated in bovine tracheal smooth muscle. We especially examined the combined effect of ibudilast with the cGMP-elevating agents on these parameters. Ibudilast was equipotent to attenuate the precontractions induced by both 0.3 microM methacholine and 40 mM K(+). By contrast, the relaxant effects of sodium nitroprusside and salbutamol on 40 mM K(+)-contracted preparations were smaller than those on 0.3 microM methacholine-contracted ones. Neither N(omega)-nitro-L-arginine (100 microM), an inhibitor of nitric oxide synthase, nor ODQ (1 H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one; 5 microM), an inhibitor of soluble guanylyl cyclase, affected the ibudilast-induced relaxation. The relaxations induced by ibudilast and diltiazem on 40 mM K(+)-contracted preparations were significantly attenuated when extracellular CaCl(2) was increased from 2.54 mM to 10 mM. Ibudilast (10 microM), which caused only minor effect by itself, significantly ( P<0.05) shifted the concentration-response curves for the relaxant responses to sodium nitroprusside (SNP), atrial natriuretic peptide (ANP) and salbutamol to the left. On the other hand, ibudilast did not change the relaxant responses to diltiazem. Unlike ibudilast, diltiazem (3 microM) failed to affect the SNP- and salbutamol-induced relaxations. Ibudilast significantly ( P<0.05) increased basal levels of cGMP and cAMP. Furthermore, ibudilast enhanced SNP (0.3 microM)- and ANP (0.3 microM)-induced cGMP accumulation and salbutamol (10 nM)-induced cAMP accumulation. Zaprinast (10 microM), a type 5 PDE inhibitor, enhanced both relaxation and cGMP accumulation induced by SNP and ANP without changing salbutamol-induced responses. These findings suggest that blockade of voltage-gated Ca(2+) channels is involved in the relaxing action of ibudilast in bovine tracheal smooth muscle. However, ibudilast potentiates relaxation responses to ANP and SNP by inhibition of PDE 5, not by blockade of Ca(2+) channels. The enhancement of cGMP-mediated response may contribute to the therapeutic effects of ibudilast.     

PDE4 and PDE5 regulate cyclic nucleotides relaxing effects in human umbilical arteries

Cyclic nucleotides (cAMP and cGMP) are the main second messengers linked to vasodilatation. They are synthesized by cyclases and degraded by different types of phosphodiesterases (PDE). The effect of PDE inhibition and cyclases stimulation on 5-hydroxytryptamine (5-HT; 1 microM) and histamine (10 microM) contracted arteries was analysed. Stimulation of guanylate cyclase or adenylate cyclase relaxed the histamine- and 5-HT-induced contractions indicating that intracellular increase of cyclic nucleotides leads to vasodilatation of the human umbilical artery. We investigated the role of different PDE families in the regulation of this effect. The presence of the different PDE types in human umbilical artery smooth muscle was analysed by RT-PCR and the expression of PDE1B, PDE3A, PDE3B, PDE4C, PDE4D and PDE5A was detected. The unspecific PDE inhibitor 3-isobutyl-1-methylxanthine (IBMX; 50 microM) relaxed histamine-contracted human umbilical artery on 47.4+/-7.2%. This effect seems to be due to PDE4 and PDE5 inhibition because among the selective PDE inhibitors used only the PDE4 inhibitor (rolipram; 1 microM) and the PDE5 inhibitors (dipyridamole and T0156; 3 microM and 1 microM respectively) induced significant relaxation (39.0+/-8.7, 30.4+/-6.0 and 36.3+/-2.8 respectively). IBMX, dipyridamole and T0156 produced similar relaxation on 5-HT-induced contraction. After forskolin, the addition of IBMX or rolipram increased the effect of the adenylate cyclase stimulator and almost completely relaxed the human umbilical artery contracted by histamine (92.5+/-4.9 and 90.9+/-4.7 respectively), suggesting a main role of PDE4. The data obtained with 5-HT contracted arteries confirmed this, because only rolipram and IBMX significantly increased the forskolin vasodilator effect. The administration of dipyridamole and T0156 after sodium nitroprusside (SNP) induced a significant increase of the SNP relaxant effect on histamine-contracted arteries, but PDE1 and PDE3 inhibition did not increase the effect of the guanylate cyclase stimulator. Similar effects were obtained in 5-HT contracted arteries, the SNP induced relaxation was increased by the PDE5 inhibition, but not by PDE1 or PDE3 inhibition. In summary, our results demonstrate that: 1) the increase of cAMP and/or cGMP levels induces relaxation of the human umbilical vascular smooth muscle; 2) four families of PDE are expressed in this smooth muscle: PDE1, PDE3, PDE4 and PDE5; 3) between these families, PDE4 and PDE5 are the key enzymes involved in the regulation of the relaxation associated to cAMP and cGMP, respectively.     

Rat atrial natriuretic factor stimulates renin release from renal cortical slices

We investigated the effect of synthetic rat atrial natriuretic factor (ANF) on renin release from rat renal cortical slices. Rat ANF (10(-6) M) increased renin release from the slices with a concomitant increase in the levels of cGMP contents. The increase in cGMP was also prominent in the case of incubation with 10(-4) M sodium nitroprusside but was not accompanied by an enhanced release renin. 8-Bromo-cGMP did not stimulate renin release. We propose that the stimulation of renin release from rat renal cortical slices is not related to an increase in endogenous cGMP.     

Diuretics modify [Arg8]vasopressin-stimulated cAMP but not atrial natriuretic peptide-stimulated cGMP formation in renal cells.

The present study was undertaken to examine whether sulfonamide-derived diuretics affect [Arg8]vasopressin (AVP)-stimulated or atrial natriuretic peptide (ANP)-stimulated cyclic nucleotide formation in cells cultured from rat or dog kidney. In rat renal cells, all four sulfonamide-derived diuretics examined significantly suppressed 10(-9) M AVP-stimulated cAMP formation at concentrations of 10(-4) and 10(-3) M, while basal cAMP formation was unchanged by the diuretics. When cells were stimulated with 10(-7) M AVP, low ceiling diuretics (indapamide and trichlormethiazide) did not suppress cAMP formation, while high ceiling diuretics (furosemide and azosemide) significantly suppressed cAMP formation at concentrations of 10(-4) and 10(-3) M. The suppressive effect of the diuretics on AVP-stimulated cAMP formation in vitro paralleled the reported diuretic potency of the agents in vivo. In dog renal cells, all four diuretics significantly suppressed 10(-9) M AVP-stimulated cAMP formation at concentrations from 10(-6) to 10(-5) M, while these diuretics did not change basal cAMP levels. High ceiling diuretics suppressed 10(-7) M AVP-stimulated cAMP formation, whereas low ceiling diuretics did not. The difference in effective doses between rats and dogs seems to be consistent with the species difference observed in vivo. None of the diuretics affected basal levels of intracellular cGMP or ANP-stimulated cGMP formation in cultured rat renal cells. In addition to the inhibition of the Na/K/Cl co-transporter, it is suggested that most sulfonamide-derived diuretics act, at least in part, by inhibiting the actions of AVP.     

Thrombospondin-1 inhibition of vascular smooth muscle cell responses occurs via modulation of both cAMP and cGMP

Nitric oxide (NO) drives pro-survival responses in vascular cells and limits platelet adhesion, enhancing blood flow and minimizing thrombosis. The matricellular protein thrombospondin-1 (TSP1), through interaction with its receptor CD47, inhibits soluble guanylyl cyclase (sGC) activation by NO in vascular cells. In vascular smooth muscle cells (VSMCs) both intracellular cGMP and cAMP regulate adhesion, contractility, proliferation, and migration. cGMP can regulate cAMP through feedback control of hydrolysis. Inhibition of the cAMP phosphodiesterase-4 selectively interfered with the ability of exogenous TSP1 to block NO-driven VSMC adhesion but not cGMP accumulation, suggesting that cAMP also contributes to VSMC regulation by TSP1. Inhibition of phosphodiesterase-4 was sufficient to elevate cAMP levels, and inhibiting guanylyl cyclase or phosphodiesterase-3, or adding exogenous TSP1 reversed this increase in cAMP. Thus, TSP1 regulates VSMC cAMP levels in part via cGMP-dependent inhibition of phosphodiesterase-3. Additionally basal cAMP levels were consistently elevated in both VSMCs and skeletal muscle from TSP1 null mice, and treating null cells with exogenous TSP1 suppressed cAMP levels to those of wild type cells. TSP1 inhibited both forskolin and isoproterenol stimulated increases in cAMP in VSMCs. TSP1 also abrogated forskolin and isoproterenol stimulated vasodilation. Consistent with its ability to directly limit adenylyl cyclase-activated vasodilation, TSP1 also limited cAMP-induced dephosphorylation of myosin light chain-2. These findings demonstrate that TSP1 limits both cGMP and cAMP signaling pathways and functional responses in VSMCs and arteries, by both phosphodiesterase-dependent cross talk between these second messengers and by inhibition of adenylyl cyclase activation.     

Antioxidants, vitamin C and dithiothreitol, activate membrane-bound guanylate cyclase in PC12 cells

Antioxidants and antioxidant enzymes are known to protect against cell death induced by reactive oxygen species. However, apart from directly quenching free radicals, little is known about the effect of antioxidants on hormone-activated second messenger systems. We previously found that antioxidants such as 17-beta estradiol and resveratrol activate membrane-bound guanylate cyclase GC-A, the receptor for atrial natriuretic factor (ANF), in PC12 cells. It is possible that other antioxidants may also activate membrane-bound guanylate cyclase GC-A. The aim of this study was to determine if dithiothreitol (DTT), vitamin C, and vitamin E activate membrane-bound guanylate cyclase GC-A in PC12 cells. The results showed that both DTT and vitamin C increased cGMP levels in PC12 cells, whereas vitamin E had no effect. DTT and vitamin C inhibited membrane-bound guanylate cyclase activity stimulated by ANF in PC12 cells. In contrast, DTT and vitamin C had no effect on soluble guanylate cyclase activity stimulated by substance P. Furthermore, NO synthase inhibitors L-NAME and aminoguanidine did not affect DTT- and vitamin C-stimulated guanylate cyclase activity. The results indicate that DTT and vitamin C, but not vitamin E, activate membrane-bound guanylate cyclase GC-A in PC12 cells.     

Inhibition of cyclic GMP hydrolysis with zaprinast reduces basal and cyclic AMP-elevated L-type calcium current in guinea-pig ventricular myocytes

(1) Cyclic GMP (cGMP) has been shown to be an important modulator of cardiac contractile function. A major component of cGMP regulation of contractility is cGMP-mediated inhibition of the cardiac calcium current (I(Ca)). An under-appreciated aspect of cyclic nucleotide signalling is hydrolysis of the cyclic nucleotide (i.e., breakdown by phosphodiesterases (PDEs)). The role of cGMP hydrolysis in regulating I(Ca) has not been studied. Thus the purpose of this study was to investigate if inhibition of cGMP hydrolysis can modulate I(Ca) in isolated guinea-pig ventricular myocytes. (2) Zaprinast, a selective inhibitor of cGMP-specific PDE (PDE5), caused a significant increase in cGMP levels in myocytes, but was without affect on basal or beta-adrenergic stimulated cAMP levels (consistent with its actions as a specific inhibitor of PDE5). (3) Zaprinast inhibited I(Ca) that was pre-stimulated with cAMP elevating agents (isoproterenol, a beta-adrenergic agonist; or forskolin, a direct activator of adenylate cyclase). The effect of zaprinast was greatly reduced by KT5823, an inhibitor of cGMP-dependent protein kinase (PKG). (4) Zaprinast also significantly inhibited basal I(Ca) when perforated-patch or whole-cell recording with physiological pipette calcium concentration (10(-7) M) was used. However, this effect was not observed when using standard calcium-free whole-cell recording conditions. (5) These results indicate that inhibition of cGMP hydrolysis can decrease both basal and cAMP-stimulated I(Ca). Thus, cGMP hydrolysis may likely be an important step for physiological modulation of I(Ca). This regulation may also be important in disease states in which cGMP production is increased and PDE5 expression is altered, such as heart failure.     

硝普钠在豚鼠离体气管螺旋条中的舒张作用

目的研究硝普钠(SNP)在豚鼠离体气管螺旋条中舒张作用的机制.方法在浴槽内加入硝普钠从10-7M至10-3M,观察气管螺旋条张力的变化,并检测浴槽中NO,气管螺旋条组织cAMP、cGMP的变化.结果加入硝普钠气管螺旋条张力下降,浴槽内NO增加,气管螺旋条组织cAMP、cGMP增加.结论硝普钠有舒张气管螺旋条的作用,推测是通过NO-cGMP系统发挥作用.     

Effects of pinacidil on serotonin-induced contractions and cyclic nucleotide levels in isolated rat aortae: comparison with nitroglycerin, minoxidil, and hydralazine

Pinacidil is a novel, clinically effective vasodilator used for the treatment of hypertension whose mechanism of action has not been precisely defined. In vitro, pinacidil (ED50 = 0.3 microM) was approximately 30-fold less potent than nitroglycerin and 700-fold more potent than minoxidil or hydralazine in relaxing rat aortic strip preparations. Aortic relaxations produced by nitroglycerin and acetylcholine were dramatically antagonized by methylene blue (10(-5) M), an inhibitor of soluble guanylate cyclase. In contrast, relaxation to hydralazine or minoxidil was unaffected and relaxation to pinacidil was only modestly inhibited (approximately threefold) by methylene blue (10(-5) M). Furthermore, aortic relaxation to pinacidil was similar in preparations with and without an intact endothelium. Relaxation induced by pinacidil (10(-7)-10(-4) M) was not associated with any elevation in either cyclic AMP (cAMP) or cyclic GMP (cGMP) levels in vitro, although nitroglycerin (10(-6) M) but not minoxidil (10(-3) M) or hydralazine (10(-3) M) significantly elevated cGMP levels. Thus, pinacidil was a potent relaxant agonist in vitro, in contrast to minoxidil and hydralazine, which were considerably weaker in this regard. Vascular relaxation produced by pinacidil was independent of an intact endothelium and was not associated with elevations in either cAMP or cGMP. These data are consistent with the proposal that the antihypertensive activity of pinacidil is due to nonspecific arterial vasodilation.     

Lack of second messenger function of cyclic GMP in acetylcholine-induced negative inotropism

As cyclic 3',5'-guanosine monophosphate (cGMP) is still discussed as a possible mediator of the negative inotropic effects of cholinergic agents, the influence of acetylcholine (ACh) on force of contraction and cGMP tissue levels was studied in isolated, electrically driven guinea pig auricles in the presence of methylene blue, an inhibitor of guanylate cyclase activation, as well as of M & B 22,948, an inhibitor of cGMP breakdown. Nitroprusside-Na (NP), a potent stimulator of guanylate cyclase, was tested for comparison under the same conditions. ACh at concentrations of 10(-7)-5 X 10(-6) M dose-dependently diminished force of contraction down to cardiac arrest, whereas NP only had a slight negative inotropic effect that was maximum at 10(-5) M and reduced force of contraction to 89% of control. Although ACh was much more effective in reducing force of contraction than NP, only NP significantly increased myocardial cGMP levels. The rise in cGMP produced by NP was attenuated by methylene blue (5 X 10(-5) M) and augmented by M & B 22,948 (3.7 X 10(-4) M), whereas the contractile effects (similar as those of ACh) remained unchanged. These results suggest that the negative inotropic action of ACh is not mediated by cGMP.     

Relaxant effects of beta-adrenoceptor agonist formoterol and BRL 37344 on bovine iris sphincter and ciliary muscle

We investigated the relaxant effect of beta2-adrenoceptor agonist formoterol and beta3-adrenoceptor agonist BRL 37344 on bovine iris sphincter and ciliary muscle and measured cAMP and cGMP levels. Iris sphincter (n = 16) and ciliary muscle (n = 16) strips were mounted in organ baths and tested for changes in isometric tension in response to formoterol and BRL 37344. Their relaxant effects on serotonin-induced contractions in the presence or absence of metoprolol, ICI 118.551 and SR 59230A (beta1-, beta2-, beta3-adrenoceptor antagonist, respectively) were investigated. Their effects on cAMP and cGMP levels in iris sphincter (n = 12) and ciliary muscle (n = 12) were evaluated. Formoterol (10(-11)-10(-5) M) and BRL 37344 (10(-10)-10(-5) M) decreased the serotonine-induced contractions in a concentration-dependent manner. Emax values of formoterol were significantly higher than those of BRL 37344 in iris sphincter and ciliary muscle, with no significant change in pD2 values. The relaxation responses by formoterol and BRL 37344 were antagonized with ICI 118.551 (10(-6) M) and SR 59230A (10(-6) M). cAMP levels of formoterol- and BRL 37344-treated tissues were significantly higher than those of the control tissues. cGMP levels of BRL 37344-treated tissues were significantly higher than those of control tissues, but this effect of BRL 37344 was less significant than its effect on cAMP levels. beta-adrenoceptor relaxation responses in bovine iris sphincter and ciliary muscle are mediated by a mixed population of beta2- and beta3-adrenoceptor subtypes, with a predominant contribution of cAMP. Potency of formoterol and BRL 37344 was similar, but efficacy of formoterol was better than BRL 37344.